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      DNA barcoding: complementing morphological identification of mosquito species in Singapore

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          Abstract

          Background

          Taxonomy that utilizes morphological characteristics has been the gold standard method to identify mosquito species. However, morphological identification is challenging when the expertise is limited and external characters are damaged because of improper specimen handling. Therefore, we explored the applicability of mitochondrial cytochrome C oxidase subunit 1 ( COI) gene-based DNA barcoding as an alternative tool to identify mosquito species. In the present study, we compared the morphological identification of mosquito specimens with their differentiation based on COI barcode, in order to establish a more reliable identification system for mosquito species found in Singapore.

          Methods

          We analysed 128 adult mosquito specimens, belonging to 45 species of 13 genera. Phylogenetic trees were constructed for Aedes, Anopheles, Culex and other genera of mosquitoes and the distinctive clustering of different species was compared with their taxonomic identity.

          Results

          The COI-based DNA barcoding achieved a 100% success rate in identifying the mosquito species. We also report COI barcode sequences of 16 mosquito species which were not available previously in sequence databases.

          Conclusions

          Our study utilised for the first time DNA barcoding to identify mosquito species in Singapore. COI-based DNA barcoding is a useful tool to complement taxonomy-based identification of mosquito species.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13071-014-0569-4) contains supplementary material, which is available to authorized users.

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          Most cited references37

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          Positional effect of single bulge nucleotide on PNA(peptide nucleic acid)/DNA hybrid stability

          Takafumi N. Sugimoto, K. Yamamoto, N Satoh (1999)
          We report positional effect of bulge nucleotide on PNA/DNA hybrid stability. CD spectra showed that PNA/DNA hybrids required at least seven base pairings at a stem region to form a bulged structure. On the other hand, DNA/DNA could form bulged structure when there are only four base pairings adjacent to the bulge nucleotide. We discuss why PNA requests such a many base pairings to form bulged structure from a nearest neighbor standpoint.
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            Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians

            Miguel Vences, Meike Thomas, Arie van der Meijden … (2005)
            Background Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI) has been proposed as universal marker for this purpose among animals. Results Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%), with low degrees of pairwise haplotype divergence within populations (0–1%). Conclusion We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.
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              Four years of DNA barcoding: current advances and prospects.

              Raphaël Leblois, Lise Frezal (2008)
              Research using cytochrome c oxidase barcoding techniques on zoological specimens was initiated by Hebert et al. [Hebert, P.D.N., Ratnasingham, S., deWaard, J.R., 2003. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B 270, S96-S99]. By March 2004, the Consortium for the Barcode of Life started to promote the use of a standardized DNA barcoding approach, consisting of identifying a specimen as belonging to a certain animal species based on a single universal marker: the DNA barcode sequence. Over the last 4 years, this approach has become increasingly popular and advances as well as limitations have clearly emerged as increasing amounts of organisms have been studied. Our purpose is to briefly expose DNA Barcode of Life principles, pros and cons, relevance and universality. The initially proposed Barcode of life framework has greatly evolved, giving rise to a flexible description of DNA barcoding and a larger range of applications.
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                Author and article information

                Contributors
                Abigail Chan: abigail_chan@nea.gov.sg
                Lee-Pei Chiang: lipasein@yodenza.com
                Hapuarachchige C Hapuarachchi: chanditha_hapuarachchi@nea.gov.sg
                Cheong-Huat Tan: tan_cheong_huat@nea.gov.sg
                Sook-Cheng Pang: pang_sook_cheng@nea.gov.sg
                Ruth Lee: ruth_lee@nea.gov.sg
                Kim-Sung Lee: lks9@np.edu.sg
                Lee-Ching Ng: ng_lee_ching@nea.gov.sg
                Sai-Gek Lam-Phua: lam-phua_sai_gek@nea.gov.sg
                Journal
                Journal ID (nlm-ta): Parasit Vectors
                Journal ID (iso-abbrev): Parasit Vectors
                Title: Parasites & Vectors
                Publisher: BioMed Central (London )
                ISSN (Electronic): 1756-3305
                Publication date (Electronic): 12 December 2014
                Publication date PMC-release: 12 December 2014
                Publication date Collection: 2014
                Volume: 7
                Issue: 1
                Electronic Location Identifier: 569
                Affiliations
                [ ]Environmental Health Institute, National Environment Agency, 11 Biopolis Way, Helios Block, #06-05/08, Singapore, 138667 Singapore
                [ ]Shizenature Pte Ltd, 3016, Eastech, Bedok North Avenue 4, #02-13, Singapore, 489947 Singapore
                [ ]School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, 535, Clementi Road, Singapore, 599489 Singapore
                Article
                Publisher ID: 569
                DOI: 10.1186/s13071-014-0569-4
                PMC ID: 4282734
                PubMed ID: 25498759
                SO-VID: dd558023-8e84-46ae-aa59-a62e03f90056
                Copyright © © Chan et al.; licensee BioMed Central. 2014
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                Date received : 27 December 2013
                Date accepted : 25 November 2014
                Categories
                Subject: Research
                Custom metadata
                issue-copyright-statement © The Author(s) 2014

                ScienceOpen disciplines: Parasitology
                Keywords: dna barcode,coi,identification,taxonomy,mosquitoes,phylogeny
                Data availability:
                ScienceOpen disciplines: Parasitology
                Keywords: dna barcode, coi, identification, taxonomy, mosquitoes, phylogeny

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